| objective: In order to Provide experimental foundation for research of DEACMP.We study the change levels of activation protein-1(AP-1)and heme oxygenase-1(HO-1)in hippocampus of rats model and explore their possible mechanisms resulting in delayed encephalopathy.Methods : Screening 156 healthy male SD rats,they are divided into three groups by using the random number table method: DEACMP group(CO,n=60),air group(AC,n=60)and blank group(BC,n=36),three groups of rats are average divided into six subgroups according to the six poisoning time of 1st day,3rd day,7th day,14 thday,21st day and 28 th day,CO group and AC group were randomly selected 10 rats at each time point,BC group selected 6 rats.Multiple intraperitoneal injection of improved CO method at intervals is used to establish DEACMP model,the same volume f air is injected in air group,while blank group without any intervention.Morris water maze experiment(divided into navigation and space exploration)is used to test groups of rats learning and memory and cognitive ability.Using HE observed the pathological changes in hippocampus CA1 area of rats.The levels of AP-1 and HO-1 are observed by immunohistochemistry,Using Western blotting method to analysis the expression of AP-1.TUNEL is used to detect apoptotic neurons in hippocampal CAl area.Results: 1.The establishment of DEACMP model: Be agitated,crashed after CO group rats infected cage,shortness of breath,no less,sleepiness,such as acute CO poisoning,the oral,toe skin mucous membrane is cherryred,poisoning serious part of rats can appear coma,convulsion and even death.AC and BC group rats are normal without the above clinical manifestations.2.Experimental results of Morris water maze: 1)the navigation test(average escape latency): the average escape latency of CO、AC、BCgroups rats in front of the canister and infected after one week had no statistically difference(P > 0.05);CO group infected after two week(21.26±3.22)and three week(23.17±3.92)and four week(22.53±2.84)the average escape latency period extended,compared with AC and BC group difference was statistically significant(P < 0.05).2)the space exploration test(platform quadrant motion time): platform quadrant motion time of three groups of rats after the time befote the virus and infected one week no statistically significant difference(P > 0.05);CO group platform quadrant motion time after infected two week(31.12±2.05)and three week(28.03±3.30)and four week(21.37±2.76)gradually shortened,compared with AC and BC group difference was statistically significant(P < 0.05).3.HE dyeing conditions: AC and BC group hippocampal CAl area at each time point the normal forms of cell neatly,cell membrane,completely boundary clear;group CO,the decrease in the number of neurons in the rats hippocampal CAl area,arrangement is very irregular,the gap between each other,the nucleus hyperchromatic,contraction and different level fracture.4.Immunohistochemical results: 1)AP-1:AC and BC group expressed less at each time point and no statistically significant difference(P > 0.05),CO group each time point,AP-1 protein expression(3.5±0.51 、7.92±0.88、6.4±1.17、5.25±1.01、4.36±1.32、3.74±0.69)were higher than that of AC and BC group,3 d to peak,group 3 d compared with the rest of the time difference was statistically significant(P < 0.05).2)HO-1:AC and BC group expressed less at each time point and no statistically significant difference(P > 0.05),CO group each time point,HO-1 protein expression(3.65±0.43 、9.06±0.86、6.86±0.57、5.00±0.77、4.2±0.72、3.72±0.59)were higher than that of AC and BC group,3 d to peak,group 3 d compared with the rest of the time difference was statistically significant(P < 0.05).5.the Western blot results of AP-1:In AC and BC group in rats hippocampal is scanty,CO group in the rat hippocampus increased when 1 d(0.93±0.07),the 3 d(1.71±0.10)when the peak,one week(1.45±0.10)began to decline,tofour week(0.65±0.10)when the expression is still higher than AC(0.39±0.04)and BC(0.50±0.07)group.The expression trend of AP-1 protein is consistent with the immunohistochemical results in CO group at each time piont.6.TUNEL staining results: AC and BC group from the subgroup rats hippocampal CAl area neurons section with a few apoptotic cells and apoptotic cells CO group was obviously higher than that of AC and BC group,the apoptosis index(7.49±0.65、15.63±1.14、20±1.56、18.22±1.00、12.41±1.19、7.86±1.01)is gradually increased,after the virus one week to the peak,then gradually decline,and infected after one week and two week respectively,compared with the rest of the time points in the group,the difference was statistically significant(P < 0.05),the poison after one week to peak.Conclusion:1.By intraperitoneal injection of multiple interval method preparation of DEACMP model of CO,a simple and safe operation,the CO less dosage,canister to depth dose the advantages of easy to control,is an ideal method to establish model of DEACMP.2.In Morris water maze experiment testing rats cognitive ability,combining with the behavior of rats HbCO level in the blood of the change and comprehensive whether CO group rats had delayed encephalopathy.3.AP-1 may by participating in regulating the expression of apoptosis related gene targets HO-1,cause neurons apoptosis after carbon monoxide poisoning,thus helping the occurrence of delayed encephalopathy. |
